Proteins have become commercially important as drugs that are also generally called “biologicals”. One of the greatest challenges is the development of cost effective and efficient processes for the production of recombinant proteins on a commercial scale.
The biotech industry makes an extensive use of mammalian cells for the manufacturing of recombinant glycoproteins for human therapy.
Today, fed-batch and perfusion cultures are the two dominant modes of industrial operation for the mammalian cell culture processes that require large amount of proteins (Hu and Aunins 1997). Whatever the production technology of choice is, development efforts aim at obtaining production processes that warrant high volumetric productivity, batch-to-batch consistency, homogenous product quality at low costs.
The decision between fed-batch or perfusion production mode is mainly dictated by the biology of the clone and the property of the product, and is done on a case-by-case basis during the course of the development of a new drug product (Kadouri and Spier 1997).
When the selection is a perfusion process, one of the culture systems of choice is stationary packed-bed bioreactor in which cells are immobilized onto solid carriers. This system is easy to operate and with appropriate carriers and culture conditions very high cell density (of ˜107-108 cell·ml−1) can be achieved.
A consequence of this high cell density is the need for an intensive medium perfusion rate (feed and harvest) that should be used in order to keep the cells viable and productive. It appears that the perfusion rate is one of the central parameters of such a process: it drives the volumetric protein productivity, the protein product quality and has a very strong impact on the overall economics of the process.
Therefore at industrial scale the optimal stationary packed-bed bioreactor process should operate with a perfusion rate as low as possible without compromising on quantity and quality of the product.
In the course of reducing the perfusion rate, several studies were conducted where the concentration of glucose (Wang et al. 2002) (Dowd et al. 2001) is used as an indicator of other nutrients level in the feed medium in order to operate the bioreactor at a low perfusion rate without accumulating in the culture high levels of toxic by-products such as lactate and ammonia (Sugiura and Kakuzaki 1998) (Racher et al. 1993). Modification of culture parameters such as pH or temperature (Chuppa et al. 1997) is also a common strategy to optimize culture conditions and reduce medium perfusion needs.
In order to achieve optimal medium perfusion rate, three approaches can be considered:
a) To fix perfusion rate at a constant value during the entire production run.
This approach is usually preferred in industrial production processes, since it is simpler to operate in a robust and consistent way. It also it has the advantage to define medium costs of the process as there is no variation in perfusion rate from run to run.
b) To adjust perfusion rate in response to cell number and/or nutrient consumption such as glucose (Oh et al. 1994) (Dowd et al. 2001) (Gorenflo et al. 2003), glutamine (Gorenflo et al. 2002), or oxygen (Kyung et al. 1994). Although this approach provides a more scientific rationale for adjusting the perfusion rate, it may lead to an overgrown culture and an “out-of-control” increase of the perfusion rate. When cells are cultured in suspension mode, a “culture bleed” is done to avoid overgrowing the culture, but this is not possible when cells are immobilized on a carrier. So in general, this approach is not preferred for manufacturing operations as it is difficult to operate in a robust and consistent manner and the medium perfusion rate needs to be readjusted on a daily basis.
c) To combine both strategies a) and b) with an initial cell propagation phase (or “growth phase”) where the perfusion rate is progressively increased according to cell growth requirements during the growth phase followed by a shift of culture conditions such as temperature and/or pH in order to stabilize and keep cell metabolism at a relatively low and constant level. At this stage, the perfusion rate can be reduced to a fixed value, matching the reduced need of the cells throughout the production phase.
It is known that modification of the perfusion rate during a perfusion process, as well as modification of other bioprocess factors, can influence the recombinant protein quality and in particular its glycosylation pattern (Jenkins et al. 1996) (Andersen et al. 2000). Glycosylation is usually recognized as an important function in the solubility, immunogenicity, and pharmacokinetic properties of human glycoproteins and those are key parameters in the safety and clinical efficacy of a product (Goochee et al. 1991). In particular, glycosylation affects folding and secretion of many glycoproteins, as well as their plasma half-life, thus having an important impact on in vivo biology and activity of glycosylated proteins.
In general, the term “glycosylation” of a protein refers to the formation of the sugar-amino acid linkage. Glycosylation is a crucial event in the biosynthesis of the carbohydrate units of (secreted) glycoproteins. It sets into motion a complex series of posttranslational enzymatic steps that lead to the formation of a host of protein-bound oligosaccharides with diverse biological functions.
Mammalian glycoproteins commonly contain three types of constituent glycans; the N-linked glycans which are attached to asparagine via an N-acetylglucosamine (GlcNAc) residue in an Asn-Xxx-(Ser, Thr) motif, where Xxx can be any amino acid except proline, those attached to serine or threonine, referred to as O-linked glycans and the carbohydrate components of glycosylphosphatidylinositol. Although many variations are possible, the antennae of mature glycans usually consist of one or more N-acetyllactosamine units with the chains terminating in either sialic acid or α-linked galactose. Fucose is frequently found attached to the asparagine-linked GlcNAc residue and often, additionally on the antennae. Other common modifications to the basic structure include a GlcNAc residue attached to the 4-position of the core branching mannose residue, referred to as a “bisecting” GlcNAc residue and sulphate groups which can be found in a variety of locations, both on the core and the antennae.
The biosynthesis of these compounds involves attachment to the asparagine of a glycan containing the trimannosyl-chitobiose core together with an additional six mannose and three glucose residues followed by removal of the glucose and four mannose residues. Various other glycosyl transferases and glycosidases then process the (GlcNAc)2(Man)5 structure to the mature glycan. This process results in three general types of N-linked glycan depending on the extent of processing; “high-mannose” glycans in which only mannose resides on the two antennae, “hybrid glycans” in which one antenna is processed and “complex” glycans where both antennae are modified. O-linked glycans, on the other hand, are much more diverse, ranging from monosaccharides to large sulphated polysaccharides with no common core structure or consensus sequence of amino acids at the attachment site (Harvey, 2001).
One such glycosylated protein of therapeutic interest is interleukin-18 binding protein.
Interleukin-18 binding protein (IL-18BP) is a naturally occurring soluble protein that was initially affinity purified, on an IL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes IL-18 induction of IFN-γ and IL-18 activation of NF-κB in vitro. In addition, IL-18BP inhibits induction of IFN-γ in mice injected with LPS.
The IL-18BP gene was localized to the human chromosome 11, and no exon coding for a transmembrane domain could be found in the 8.3 kb genomic sequence comprising the IL-18BP gene. Four isoforms of IL-18BP generated by alternative mRNA splicing have been identified in humans so far. They were designated IL-18BP a, b, c, and d, all sharing the same N-terminus and differing in the C-terminus (Novick et al 1999). These isoforms vary in their ability to bind IL-18 (Kim et al. 2000). Of the four human IL-18BP (hIL-18BP) isoforms, isoforms a and c are known to have a neutralizing capacity for IL-18. The most abundant IL-18BP isoform, isoform a, exhibits a high affinity for IL-18 with a rapid on-rate and a slow off-rate, and a dissociation constant (Kd) of approximately 0.4 nM (Kim et al. 2000).
IL-18BP belongs to the immunoglobulin superfamily.
The residues involved in the interaction of IL-18 with IL-18BP have been described through the use of computer modelling (Kim et al. 2000) and based on the interaction between the similar protein IL-1β with the IL-1R type I (Vigers et al. 1997).
IL-18BP is constitutively present in many cells (Puren et al. 1999) and circulates in healthy humans, representing a unique phenomenon in cytokine biology. Due to the high affinity of IL-18BP to IL-18 (Kd=0.4 nM) as well as the high concentration of IL-18BP found in the circulation (20 fold molar excess over IL-18), it has been speculated that most, if not all of the IL-18 molecules in the circulation are bound to IL-18BP. Thus, the circulating IL-18BP that competes with cell surface receptors for IL-18 may act as a natural anti-inflammatory and an immunosuppressive molecule.
IL-18BP has been suggested as a therapeutic protein in a number of diseases and disorders, such as psoriasis, Crohn's Disease, rheumatoid arthritis, psoriatic arthritis, liver injury, sepsis, atherosclerosis, ischemic heart diseases, allergies, etc., see e.g. WO9909063, WO0107480, WO0162285, WO0185201, WO02060479, WO02096456, WO03080104, WO02092008, WO02101049, WO03013577.
The prior art does not describe a process for the production of recombinant IL-18BP in CHO cells, nor IL-18BP compositions characterized by a specific glycosylation profile.